Nowadays, herbal products, especially their polyphenolic compounds, are intensely studied due to their high radical scavenging and antioxidant activities ⁵. Of them is the pomegranate. The juice and other compounds of pomegranate are the rich sources with respect to vitamin C, minerals, and polyphenols ⁶ that possess high antioxidant activity ⁷. The other herbal product, cinnamon, has been used as a spice and also treatment of many diseases for traditional medicine. Cinnamomum zeylanicum, C. cassia and C. camphora are the most important species of cinnamon in terms of obtaining the volatile oils ⁸. Eugenol and cinnamaldehyde are the main chemical compounds of oils extracted from the leaves and barks of C. zeylanicum, respetively ^8-9. High free radical scavenging and antioxidant activities of different cinnamon extracts have recently been demonstrated in many studies ^10-15. The hepatoprotective effects of cinnamon on acute liver damage were reported by some investigators ^12,13,16. However, there is no study regarding the protectiveness of cinnamon bark oil (CBO) on long term liver damage, apoptosis and tissue oxidative stress induced by CCl4 in rats.

In this study, the probable hepatoprotective effects of pomegranate juice (PJ) and CBO consumption on CCl4-induced chronic damages were investigated by examining the changes in histopathological structure, apoptotic cells and oxidant-antioxidant balance in liver tissues of rats.

2. Animals and study design: The approval of Animal Experimentations Local Ethics Committee of Firat University (Elazig, Turkey) was taken for the animal use and study design. Fourty-two healthy adult male Wistar albino rats, at the age of 5 months, were obtained from Firat University Experimental Research Centre (Elazig, Turkey) and the study was maintained therein. Standard laboratory conditions (12 h day / 12 h night, 24±3 °C temperature, 45% to 65% humidity) were provided. The balanced commercial diet (Elazig Food Company, Elazig, Turkey) and drinking water were given ad libitum during the study period.

The rats were randomly divided into six groups, 7 rats each. Control group received 0.5 mL pure olive oil + 1.5 mL distilled water daily; PJ group was administered with 5 mL/kg PJ (approximately 1.5 mL volume for each rat) + 0.5 mL pure olive oil daily; CBO group was given with 0.5 mL of olive oil containing 100 mg/kg CBO+1.5 mL distilled water daily; the rats in CCl4 group were treated with 0.25 mL/kg CCl4, which dissolved in 0.5 mL pure olive oil, weekly+1.5 mL distilled water daily; the rats in CCl4+PJ group received weekly CCl4 and daily PJ; and the rats in CCl4+CBO group were administered weekly CCl4 and daily CBO. All applications were done by intra-gastric tube and maintained for 10 weeks. CCl4 was dissolved in olive oil because it is a water-insoluble chemical. The administration doses of CCl4, PJ and CBO used in the present study were determined according to the earlier studies ^15,17. Each rat was weighed weekly and the amounts of CCl4, CBO and PJ were adjusted based on the alterations observed in body weights during the study period.

3. Collection and preparation of liver tissues: Ether anesthesia was used to sacrifice the rats at the end of the study. The liver samples were taken and protected against light. The half of tissue was fixed in formalin solution for histopathological examinations. The other half of tissue samples were washed three times in cold isotonic saline (0.9% v/w) and stored at −20 °C until being analyzed for oxidative stress indicators. For the determination of oxidative stress indicators, the liver tissues were minced on glass and homogenized by a glass–glass homogenizator in cold physiological saline on ice. Then, the tissues were diluted with a 9-fold volume of phosphate buffer (pH 7.4).

4. Measurement of liver oxidative stress indicators: The spectrophotometer (Shimadzu 2R/UV-visible, Tokyo, Japan) was used for the measurements of all oxidative stress indicators. Lipid peroxidation (LPO) level was measured according to the concentration of thiobarbituric acid reactive substances and the amount of malondialdehyde (MDA) produced was used as an index of LPO. The MDA level at 532 nm was expressed as nmol / g protein ¹⁸.

Reduced glutathione (GSH) level was measured using the method described by Sedlak and Lindsay ¹⁹. The level of GSH at 412 nm was expressed as nmol / g protein. Glutathione-peroxidase (GSH-Px, EC 1.11.1.9) activity was determined according to the method described by Lawrence and Burk ²⁰. The GSH-Px activity at 340 nm was expressed as IU/g protein. Catalase (CAT, EC 1.11.1.6) activity was determined by measuring the decomposition of hydrogen peroxide (H2O2) at 240 nm and was expressed as k/g protein, where k is the first-order rate constant ²¹. Protein concentration was determined using the method of Lowry et al ²².

5. Histopathological examination: The liver specimens were embedded in paraffin for histopathological examination and taken 5 μ serial sections. The sections were stained with hematoxylin and eosin (HE) and Masson's trichrome stain (MT) for evaluation of the fibrosis. The histopathological analyses were performed unawares from group separations by light microscopy. The grading of liver damage was made four severity grades were used as: 0 (none), no fibrosis and normal liver architecture; + (mild), fatty degenerations around portal areas, increased fibrosis in portal areas and sinusoidal space, and regular liver architecture; ++ (moderate), thin fibrous septa seen frequently in connecting portal areas and pseudolobules; +++ (severe), thick fibrous septa and collagen bands accompanied by pseudolobules. Also ten different periportal sites in liver sections stained with MT in each animal were calculated with computer software as the percent ratio of fibrotic areas in liver parenchym.

6. Determination of apoptotic cells in the liver: Apoptosis was detected by the terminal deoxynucleotidyl transferase (Tdt)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labeling (TUNEL) method by using an in situ cell death detection kit with horse radish peroxidase (POD) (Roche Diagnostics, Cat No: 11 684 817 910).

In order to determine the apoptotic index of hepatocytes; randomly selected ten different areas of seven sections per group were evaluated at x 400 magnification for the analysis of TUNEL staining. The apoptotoic index of hepatocytes was determined as the percentage of TUNEL positive cells with respect to the total number of cells counted using the formula: Apoptotic index = (Number of positive cells/Total number of cells counted) x100 ²³.

7. Statistical analysis: The SPSS/PC (Version 21.0; SPSS, Chicago, IL) program was used for the analysis of the all parameters. The values are presented as mean±SEM. P< 0.05 was accepted as significance. The differences between the groups with respect to all parameters were detected by using one-way analyses of variance (ANOVA). Tukey-HSD test was used for multiple comparisons.

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Tablo 1: Liver weight and liver index in control and treatment groups

2. Changes in the liver tissue oxidative stress indicators: The values of oxidative stress indicators are given in Table 2. The liver MDA levels were significantly (P<0.01) higher in CCl4 group than the control, PJ and CBO groups. PJ and CBO administration to the CCl4-treated rats significantly (P<0.01) decreased the MDA levels in tissue when compared to the CCl4 alone group. CCl4 treatment caused significant (P<0.05) decreases in the GSH-Px activities of liver in comparison to the control group. Administration of PJ and CBO to CCl4-treated rats prevented the CCl4-induced decreases in GSH-Px activities (P<0.05). With respect to liver MDA level and GSH-Px activity, there was no significant difference among control, PJ and CBO groups. Liver GSH levels were significantly (P<0.05) lower in the CCl4 group than that in the control group. However, a significant (P<0.05) increase was observed in the GSH level of CCl4+PJ and CCl4+CBO groups when compared to CCl4 group. While the significant decreases were determined in the CAT activities of liver (P<0.001) in CCl4 group when compared to the control group, the administrations of PJ and CBO to CCl4-treated rats significantly increased the decreased CAT activities in liver (P<0.001). There was a significant difference between the CBO and the PJ groups in terms of CAT activity (P<0.001). However, no statistical differences were observed in the CAT level of CCl4+PJ group when compared to CCl4+CBO group.

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Tablo 2: Malondialdehyde (MDA) and reduced glutathione (GSH) levels, glutathione-peroxidase (GSH-Px) and catalase (CAT) activities in liver tissues of rats in control and treatment groups

3. Histopathological findings in the liver: The histopathological changes observed in liver tissue are presented in Table 3. There was no abnormal appearance or histological changes in the liver of control, PJ and CBO treated rats, of which hepatic cells well preserved with cytoplasm and had prominent nucleus, nucleolus and visible central veins (Figure 1A, B, C) Mild sinusoidal congestion was observed only in PJ- and CBO-treated groups. In addition, insignificant periportal cell infiltration and biliar duct proliferation were found only in CBO-treated group. However, marked hepatic damage was found in CCl4 group. Periportal, periaciner and postnecrotic fibrosis, bile duct hyperplasie and oval cell proliferation, macrovesicular fatty change were the most frequent and distinctive findings in the liver samples in CCl4-treated rats. Especially at periportal area, increasing fibrous connective tissue spread to liver parenchyma related to regenerative nodule formations in this group (Figure 1D). Mononuclear cell infiltrations in all fibrotic areas, especially in periportal areas, were present. In addition, significant Kupffer cell activations were observed in the periportal region. Along with hydropic degeneration, cellular necrosis, karyomegaly, intranuclear inclusion globules, and acini formations were noticed in hepatocytes. It was also observed that the periportal zone of hepatocytes contained two nuclei. Free or intracytoplasmic accumulation of dark yellow bile pigment was present in the periportal hepatocytes.

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Figure 1: Normal appearance of the livers in control (A), PJ (B) and CBO (C) groups, D. Appearance of postnecrotic fibrosis, bile duct hyperplasie (arrow heads), fatty degeneration, regenerative nodule formation, mononuclear cell infiltration, karyomegaly (thick arrow), accumulation of bile pigment (Thin arrows) in rat liver treated with CCl4 for 10 weeks, E. Mild fibrosis, ovalocyte proliferation (arrow head) and karyomegaly (arrows) in CCl4+PJ group F. Mild fibrosis, intranuclear inclusion globules (arrow) in CCl4+CBO group (H&E x100).

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Table 3: The existence and severity of some pathological lesions, percent of fibrotic areas in periportal region and apoptotic hepatocyte index in liver tissues of rats in control and treatment groups

PJ or CBO consumption by CCl4-treated rats was observed to decrease the CCl4-induced the severity of all hepatic lesions except bile duct hyperplasia, hydropic degeneration in hepatocytes and periportal cell infiltrations (Figure 1E, F). Severity of periportal infiltrations was lesser in CCl4+PJ, but not in CCl4+CBO, when compared to alone CCl4 group. Control (Figure 2A), PJ- (Figure 2B) and CBO-treated rat livers (Figure 2C) were similar, and they had also normal levels in terms of fibrous tissue in periportal areas when compared to CCl4, CCl4+PJ and CCl4+CBO groups (Table 3). The fibrous tissue formations in periportal areas were determined as 71% in liver tissue stained with MT in CCl4 group (Figure 2D). However, PJ and CBO consumption by CCl4-treated rats significantly reduced (P<0.001) the severity of periportal fibrosis at the rates of 43% and 51%, respectively when compared to alone CCl4-treated rats (Figure 2E, F).

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Figure 2: Normal appearance of the livers in control (A), PJ (B) and CBO (C) groups, D. Appearance of severe fibrosis in rat liver in treated with CCl4 for 10 weeks, E. Mild fibrosis in CCl4+PJ group F. Mild fibrosis in CCl4+CBO group (MT x50).

4. TUNEL findings: Apoptotic cell index in hepatocytes is presented in Table 3. Figures 3A, 3B and 3C illustrate apoptosis, demonstrated by TUNEL-staining, in the liver of control, alone PJ- and CBO-treated groups, respectively. No significant differences were detected among the control, PJ and CBO groups with respect to TUNEL positivity. The apoptotic cell index of CCl4 group (Figure 3D) was significantly (P<0.001) higher than that in the control, PJ and CBO groups. However, a significant (P<0.001) decrease was observed in apoptotic cell index of CCl4+PJ (Figure 3E) and CCl4+CBO (Figure 3F) groups in comparison to only CCl4 group. No significant difference was observed in TUNEL positivity between the CCl4+PJ and CCl4+CBO groups.

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Figure 3: TUNEL positivity in control (A), PJ (B) and CBO (C) groups, D. Marked increase in TUNEL+ staining in hepatocytes of CCl4-treated group compared to the control, E. Decrease in TUNEL+ staining in hepatocytes of CCl4+PJ group, F. Decrease in TUNEL+ staining in hepatocytes of CCl4+CBO group (MT x200).

The main non-enzymatic antioxidant of cells is the GSH that involves in the metabolism of numerous toxic agents. GSH may block LPO by preventing free radical overproduction and by preserving cytochrome P450 ³⁶. In this study, CCl4 administration led to a significant decrease in the GSH level which can be an important factor in the CCl4 toxicity. However, long-term PJ and CBO administration to CCl4-treated rats significantly increased the hepatic GSH levels. It has been reported that the mechanism of hepatoprotection by PJ aganist CCl4 toxicity might be due to restoration of the GSH level ³⁰ which is an agreement with our findings. These findings were also supported by the histopathological and TUNEL analyses of liver tissues in groups treated with PJ and CBO in the present study.

CAT and GSH-Px are the other cellular antioxidants and they catalyse H2O2 to water. In this study, CCl4 administration declined the activity of the antioxidant enzymes in liver, which is confirmed by previous study ³⁷. PJ having different classes of polyphenols and flavonoids has been shown to prevent the reductions in GSH-Px and CAT acitivities by its positive effect on antioxidant enzyme activities in vivo ^30,38. In addition, C. zeylanicum ethanolic extract ¹⁶ and C. vernum ethanolic or aqueous extract ¹³ consumption has been reported to improve significantly antioxidant enzyme activities in CCl4-treated rats. Treatment of rats with CCl4 significantly reduced the activities of liver GSH-Px and CAT at the rates of 81% and 70%, respectively in this study. However, treatment of the rats with the PJ and CBO prevented the CCl4-induced decreases in GSH-Px and CAT activities, which are comparable with the control values in the present study. This shows the protection provided by PJ and CBO consumption by rats depending on maintaining the levels of these enzymes even after CCl4 treatment.

It has been reported that oxidative stress is one of the pathologic mechanisms of hepatic fibrosis ³⁹. The increased MDA levels result from the reaction between free radicals and polyunsaturated fatty acids under high oxidative stress ⁴⁰ increase the collagen synthesis ^39,41,42. The excessive levels of reactive oxygen species generated by CCl4-damaged liver cells have been reported to affect stellate cell activation and to enhance the production of the extracellular matrix, containing collagen ⁴³. The antioxidant substances within the PJ have been alleged to decline the amount of connective tissue in liver by means of their positive effects on down-regulation of excessive expression of fibrogenic cytokines and collagenes ⁴⁴. Flavonoids have counteractive effect on collagen accumulation that is the early stage of the fibrotic process ^45, and they increase the activity of protective enzymes ^46,47. It has been postulated that the beneficial strategies are the activation of antioxidase and the inhibition of LPO for preventing of early stage hepatic fibrogenesis ⁴⁸. CCl4 has been reported to cause necrosis ^28, fibrosis ^30, inflammatory cell infiltration ^49, fatty accumulation and degeneration of hepatocytes ^16,49, increases in mitotic activity ⁵⁰ and cirrhosis ⁵¹ in liver. Aforementioned histopathological lesions determined by different researchers are exactly similar to our findings found in the liver tissue after CCl4 treatment. Besides, in the present study, the CCl4-induced histopathological findings were significantly decreased by PJ and CBO treatment. The protective effect exerted by the PJ ^28,30 and CBO ¹⁶ against CCl4-induced liver damage was confirmed by conventional histological examination. In the present study, CCl4 treatment caused ~8-fold increase in periportal fibrosis as compared to control. However, hepatic tissues of CCl4+PJ and CCl4+CBO groups showed consistent reduction in the liver fibrosis and the cirrhotic process. Especially, Masson's trichrome staining showed more regular liver architecture, in which only thin fibrous bands connecting portal areas were seen in CCl4+PJ and CCl4+CBO treatment groups compared to alone CCl4 group.

It has been reported that CCl4 causes apoptosis in liver ^30,50,52. Apoptosis is the first step lesion observed in the hepatic tissue. Apoptosis and proinflammatory cytokine genes, which are inhibited during the engulfing of apoptotic bodies by macrophages ^53, have been suggested to involve in liver fibrosis induced by CCl4 ^54,55. It has been reported that CCl4 substantially affect the cytokine-related genes ⁵⁶. In addition, DNA fragmentation along with oxidative stress has been reported to play a crucial role in CCl4-induced hepatotoxicity leading increased apoptotic liver cells ⁵⁷. There was an about 12-fold increase in apoptosis in CCl4 group in comparison to the control group in this study. However, we found that PJ and CBO consumption markedly decreased the number of TUNEL-positive cells in rats treated with CCl4. In the present study, increased LPO induced by decreased CYP activity after CCl4 administration might have possibly caused the liver histopathological damages and increase in the liver apoptotic index.

It was concluded that two herbal antioxidants, PJ and CBO, have ability to prevent the CCl4 damages in liver tissue rats. The hepatoprotective effects of PJ and CBO are possibly due to the reductions in liver fibrogenesis and apoptosis associated with the prevention of CCl4-induced oxidative stress. These actions of PJ and CBO are of significant clinical importance in the prevention of alcohol abusing- or other xenobiotics-induced liver damages show similarity to the CCl4-intoxication model.

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